This proposal focuses on the mechanism(s) underlying the impact of genetic variation on the expression of genes critical for the pathogenesis of human allergic inflammation. This process, characterized by a profound dysregulation of T helper (Th)2 responses and increased expression of Th2 cytokines (IL4, IL13, IL5), has a strong genetic component, eloquently illustrated by association studies. The current challenge is to identify the mechanisms through which natural genetic variation influences gene expression and disease susceptibility. Our model gene is IL13, a key effector of allergic inflammation. Genetic variation in IL13 is strongly associated with susceptibility to asthma/allergy in ethnically diverse populations. During the previous funding cycle we showed that /L73+2044GA, a single nucleotide polymorphism (SNP) in the IL13 coding region, results in the expression of a variant (IL13 R130Q) with increased biological activity, whereas /L73-1112CT, a SNP in the IL13 promoter, leads to increased IL13 transcription in polarized Th2 cells. Furthermore, we generated a high resolution map of DNase I hypersensitive (HS) sites in the IL13 locus of neonatal CD4+ Th cells, naive and polarized under Th1 and Th2 conditions, and we identified patterns of chromatin accessibility associated with Th differentiation. Of note, several HS sites map, or are adjacent to, SNPs. In this application we use these results as a springboard to launch new approaches that will allow us to define the impact of genetic variation on mechanisms altering chromatin structural dynamics. Our overall hypothesis is that natural genetic variation within, or in the vicinity of, functional HS sites in the IL13 locus may affect the regulatory properties of these elements and alter the regulation of lL13 expression in CD4+ Th cells. To test our hypothesis, we will: Specific Aim 1: Determine whether and how genetic variation in functional IL13 HS sites affects their regulatory properties. We will analyze the role of HS sites in the in vivo regulation oflL13 expression at the level of chromatin and transcription using constructs stably integrated in CD4+ Th1 and Th2 cells, and we will assess whether and how polymorphisms affect the regulatory functions of these HS elements. Specific Aim 2: Analyze long-range intrachromosomal interactions involved in the regulation of lL13 expression and determine the impact of genetic variation. We will extend mapping of DNase I HS sites to include the entire Th2 cytokine locus and identify distal cis-regulatory element(s) engaged in long-range interactions with the IL13 locus using chromosome conformation capture (3C) and human primary Th cells. Furthermore, we will assess whether such interactions are affected by polymorphisms in IL13 HS sites. These studies will define fundamental mechanisms of IL13 regulation and provide a novel perspective on the mechanisms underlying the impact that natural genetic variation within, or in the vicinity of, regulatory elements has on the function of these elements.